By: John Quinn, PhD
A fun perk of working as a FlowJo, LLC application scientist is getting a tour of the lab I’ve visited for training. As an admitted cytometry nerd, I enjoy getting to see new technology, to hear how a given acquisition machine or probe is working out, or to see the new upper-floor lab space allocated to the cytometry core. I get to do this a decent amount, because you love showing off your hard-earned new tools as much as I enjoy seeing them. Recently, a trend that’s hard to miss is widespread adoption of the new CytoFlex cytometer by Beckman Coulter. CytoFlex users are inquiring in droves about how the 7-decade data will work in FlowJo™, so it’s time for a Daily Dongle post.
First off, FlowJoTM reads any FCS file. FCS stands for Flow Cytometric Standard—the industry rules for exporting cytometer data. Currently, all cytometer manufacturers do a good job of following these rules when they create data files, so FlowJoTM can read the data from any cytometer. If you’re interested in reading about those rules, they are available on our documentation website and ISAC’s site.
A feature of FlowJoTM version 10 is cytometer-based preferences. Previous versions of FlowJoTM included a single scaling preference that was applied to all data. In version 10, cytometer-based preferences allow FlowJoTM to read the $CYT keyword that identifies the specific cytometer the data came from, and apply the correct scaling preferences for that machine. We’ve worked with the instrument manufacturers to determine the correct preference settings to make the data look as it appears on the acquisition software and coded those preferences into FlowJoTM. We only display the preferences for machines that you’ve actually added data from, but they’re all there. Because these are preferences, you can adjust them to suit your data. To see or set the cytometer preferences:
- Open the preferences using the heart icon:
The CytoFlex is new, so we’re still optimizing its preference settings. Currently, if you load CytoFlex data into FlowJoTM using the default scaling settings, you’ll get linear and log scale graphics that look like these screen shots:
Working with Melis McHenry of Beckman Coulter, we found an improved set of preferences (which are also visible in the figure above, displaying my preferences):
- Log max = 10,000,000
- Linear max = 1,500,000
- Biexponential enabled
- Width basis (our term for the range of the linear portion of the data) = –250
With the new preference settings applied, the data from the default scale plots above look like the plots below. These are completely ungated data, hence the noise in the plots.
We’ll continue to improve the default, and are looking into how baseline subtraction interacts with FlowJoTM to make your experience seamless. In the meantime, if you have any questions you can contact either Melis or myself for help.